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K Muralidhar

Publications and source records attributed to K Muralidhar.

At least 19 recordsLinked to original sources
PubMed2006

Molecular dissection of an hCG-beta epitope using single-step solid phase radioimmunoassay.

BACKGROUND: Peptides and proteins have both sequence-specific (contiguous) and conformation-specific (discontiguous) epitopes. Sequence-specific epitopes are delineated by peptide approach and other robust methods like competition assays, gene expression assays, synthetic peptide library based assays etc. Available methods for delineation of conformation-specific epitopes are cumbersome (X-ray crystallography etc.), time-consuming and require costly sophisticated equipments. Hence, there is a need to develop a simple method for identification and mapping of conformation-specific epitopes. METHOD: In the single-step solid phase radioimmunoassay (SS-SPRIA), an immunochemical bridge of 'mouse IgG-anti-mouse IgG' was prepared in the polypropylene wells followed by adsorption with hCG specific monoclonal antibody (MAb) G(1)G(10).1. The extent of competitive inhibition in binding ability of (125)IhCG-beta with chemically or enzymatically modified hCG-beta to immobilized MAb G(1)G(10).1 in comparison to hCG-beta standards was utilized to identify the epitopic amino acid involved in epitope-paratope interaction. RESULTS: Data clearly suggest that the epitope under investigation consisted of Arg (94, 95) and Asp (99) at the core region with a Lys (104) and a His (106) in the proximity and absence of chymotrypsin susceptible Phe or Tyr in this region. CONCLUSION: The data of SS-SPRIA revealed the 93-100 loop of amino acid sequence, as the core region of conformation-specific epitope of hCG-beta at or near the receptor-binding region. Hence, SS-SPRIA seems to be a simple method for identification and mapping of conformation-specific epitopes.

Amino Acid Sequence↗
PubMed2005

Reconstruction of the concentration field around a growing KDP crystal with schlieren tomography.

Salt concentration distribution around a potassium dihydrogen phosphate (KDP) crystal growing from its aqueous solution has been experimentally determined using a laser schlieren technique. The growth process is initiated by inserting a KDP seed into its supersaturated solution, followed by slow cooling of the solution. Fluid convection leads to a distribution of concentration around the growing crystal. The pattern and strength of convection are important factors for the determination of the crystal growth rate and quality. Experiments have been conducted in a beaker with a diameter of 16.5 cm and a height of 23 cm. A monochrome schlieren technique has been employed to image the concentration field from four view angles, namely, 0 degrees, 45 degrees, 90 degrees, and 135 degrees. By interpreting the schlieren images as projection data of the solute concentration, the three-dimensional concentration field around the crystal has been determined using the convolution backprojection algorithm. The suitability of the overall approach has been validated using a simulated convective field in a circular differentially heated fluid layer, where full as well as partial data are available. Experiments have been conducted in the convection-dominated regime of crystal growth. The noncircular shape of the crystal is seen to affect axisymmetry of the concentration field close to the crystal surface. The reconstructed concentration fields reveal symmetry of the flow field away from the growing crystal. The solute concentration contours show large growth rates of the side faces of the crystal in comparison with the horizontal faces. In this respect, the concentration profiles are seen to correlate with the crystal geometry.

Journal Article↗
PubMed2005

Enhanced yield and homogeneity of buffalo growth hormone by an improved chromatographic protocol.

Loss of buffalo Growth Hormone (buGH) in the various side fractions of standard buGH purification protocol has been determined quantitatively by direct binding ELISA and qualitatively by SDS-PAGE and Western blot analysis. Accounting result indicated that there was a considerable loss of buGH in the side fractions. An alternative protocol to prevent loss and to obtain a high yield of buGH has been developed by introducing anion exchange chromatography, QAE-Sephadex. This has resulted in a simple, reproducible three-step protocol. In this protocol, an extract obtained at 250 mM (NH4)2 SO4, pH 5.5, was loaded onto the QAE-Sephadex column in 0.1 M NH4 HCO3. At this salt concentration, the bulk of the buGH came as QAE unbound fraction. Some amount of buGH, together with contaminating proteins, was bound to QAE-Sephadex and these could be eluted with 1 M KCl. The immunopotency of the enriched buGH preparation "QUB" (QAE unbound fraction) in a direct binding ELISA was similar to that of the semi-pure buGH (ECS/APECS) preparation obtained using the standard protocol, but the yield was 4 times higher. The SDS-PAGE data showed that the banding pattern of standard semi-pure buGH and QUB were quite similar and QUB can be loaded onto the Sephacryl S-200 gel filtration chromatography to yield a highly purified buGH. SDS-PAGE and Western blot analyses showed the major band of buGH in QUB at the same position as in the case of standard buGH. It has also been demonstrated here that it is possible to separate buffalo prolactin (buPRL) and buGH on QAE-Sephadex.

Animals↗
PubMed2005

A reference preparation of buffalo pituitary follicle stimulating hormone using lectin affinity chromatography.

An improved and cost effective method to isolate FSH from buffalo pituitary glands is described here. The buFSH activity was monitored throughout by a highly sensitive heterologous radioimmunoassay (sensitivity 0.2 ng oFSH/mL) and the in vivo biological activity of the final preparation was also established. A biologically active buFSH-enriched preparation with a moderate recovery (42%) was obtained. The yield of the final buFSH-enriched preparation was 26.5 mg/kg of buffalo pituitary gland. In SDS-PAGE, the purified buFSH resolved as a heterodimer of 30 kDa molecular size, with a 21 kDa presumptive alpha-subunit. This preparation was also characterized in terms of biological and some of its physicochemical properties. A high-titer antiserum to buFSH was also raised in rabbit using this preparation. The reagents generated, buFSH and buFSH-specific polyclonal antisera, have possible diagnostic and therapeutic usage for improvement of reproductive health of water buffaloes.

Animals↗
PubMed2005

Isolation of hCG and its characterization by radioimmunoassay, enzyme-immunoassay, and radio-receptor assay.

The nature of human chorionic gonadotropin (hCG) molecules present during early pregnancy of Indian women is poorly understood. Therefore, a study has been undertaken to isolate hCG and characterize different forms of hCG from urine. The hCG molecules from urine of pregnant women (45-75 days post LMP) were adsorbed onto kaolin, eluted with ammonium hydroxide, and precipitated using acetone and then lyophilized. The lyophilized extract was subjected to Sephadex G-100 chromatography followed by ion-exchange fractionation. Three major fractions of protein (i.e., Peaks I, II, and III) associated with carbohydrate activity were obtained. Peaks II and III eventually resolved into a single peak I following repeated ion exchange chromatography, which suggested the presence of aggregates of molecules. Further purification on an affinity column resolved all three peak fractions into one unadsorbed and two adsorbed (A and B) fractions. These adsorbed fractions were characterized by radioreceptor assay (RRA), radioimmunoassay (RIA), and enzyme linked immunosorbent assay (ELISA). The activity was standardized against WHO reference preparation 75/589. Peaks I (A and B) were found to have maximum at about 75% of immunologically potent hCG, followed by peaks II (40%) and III (5%). The molecular sizes of peaks I, II, and III on a Sephadex G-200 column corresponded to 27,500D, 66,000D, and 84,000D, respectively. Relative mobilities of all adsorbed fractions in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed the presence of hCG-alpha (mol. wt. 19,539D) and hCG-beta (28,870D) subunits. The presence of both subunits of hCG were also revealed by Western blot analysis. For the first time, we report the low molecular weight hCG molecule, of 27,500D by size exclusion chromatography, which has immunological and biological activity as measured by RIA, ELISA, and RRA.

Blotting, Western↗
PubMed2005

Isolation of alpha- and beta-subunits of peak-I hCG and generation of highly specific polyclonal antisera.

Development of polyclonal antisera is still a choice in some hard-pressed budget laboratories. In the present study, an attempt was made to isolate alpha- and beta-subunits from peak-I hCG, generation of polyclonal antisera and their characterization. The anti-hCG-a antisera showed titres of 1: 8000 and anti-hCG-beta antisera 1: 16,000 at 50% binding to radiolabelled hCG in RIA. Studies on specificity using anti hCG-beta antiserum demonstrated no cross-reaction with several hormones tested in the present study, except for hCG-beta and hCG, thus eliciting a highly specific hCG-beta antiserum.

Animals↗
PubMed2004

Use of SP-Sephadexto fractionate and obtain semi-pure preparation from whole pituitaries of Indian water buffaloes (Bubalus bubalis).

Different charge isoforms of native Luteinizing hormone (LH) (dimeric form) Ve/Vo = 1.49 can be fractionated on SP-Sephadex into four different charge isoforms (LHUB, LH25, LH50, and LH100) by stepwise elution using different molarities of Na2HPO4. LHUB was found to be difficult to purify, whereas LH50 and LH100 were found to be pure and highly immunoreactive against anti-bLHbeta serum as indicated by the results obtained from direct binding ELISA and Western blot analysis. SDS-PAGE of LH50 and LH100 showed two bands corresponding to the two subunits of LH. LH25 can be purified to homogeneity by rechromatography on S-300. Purification of buLH as a highly immunoreactive preparation has also been described using SP-Sephadex. This LH preparation (SP25B), which was obtained after slight modification in the pre-existing protocol, has been found to be highly immunoreactive against anti-bLHbeta serum in direct binding ELISA. Being a very simple and reproducible method, it can be used to obtain pure LH preparation, as a reference material, in a short period of time for various immunoassays and bioassays.

Animals↗
PubMed2004

Simultaneous isolation of prolactin and growth hormone from "discarded acid pellet" obtained from buffalo pituitaries.

An acid pellet, obtained as a side fraction from a conventional gonadotropin purification pathway, has been found to contain the bulk of the pituitary lactogenic hormones (growth hormone or GH and prolactin or PRL). This discarded side fraction has been utilized to obtain buffalo lactogenic hormones (buGH and buPRL), simultaneously, and in bulk. The immunoreactivities of the purified semi-pure buffalo GH and PRL (APECS and APP-I, respectively) preparations were compared by direct binding ELISA with semi-pure standard buGH and PRL (ECS and EP-I, respectively) and were found to be as pure as standard semi-pure buGH and buPRL. When checked by direct binding ELISA using buGH and buPRL antisera, it was observed that APECS and APP-I were not cross-immunoreactive. SDS-PAGE and western blot analysis of APECS and APP-I showed major bands located at the same positions as in the case of standard semi-pure preparations (20 kDa for APECS and 23 kDa for APP-I). The semi-purified buGH and buPRL (APECS and APP-I) were converted to a highly purified preparation by chromatographing them via Sephacryl S-200 gel-filtration chromatography.

Animals↗
PubMed2004

Improved targeting of photosensitizers by intratumoral administration of immunoconjugates.

Biodistribution of technetium (99mTc) labeled hematoporphyrin derivative (HpD, Photosan-3) conjugated to a monoclonal antibody to carcinoembryonic antigen (anti-CEA) was compared following intravenous (i.v.) and intratumoral (i.t.) administration in solid Ehrlich ascites tumor bearing mice. Images of mice at different time intervals were acquired after injection of radiolabeled PS-3 in either conjugated or unconjugated forms. Quantitative estimation of the radiolabel in different tissues was performed by selecting the different region of interests (ROIs). Maximum accumulation of both free and antibody conjugated PS-3 following i.v. administration was observed in liver followed by tumor. Tumor/muscle (T/N) ratio was more with free PS-3 compared to conjugated PS-3. Pharmacokinetics of free and conjugated PS-3 was also different with faster accumulation of conjugated PS-3 in the tumor. With intratumoral administration of anti-CEA-PS-3-99mTc, specific accumulation and retention of the sensitizer was observed in the tumor tissue. Since, direct injection of antibody conjugated photosensitizer into the tumor resulted in longer retention of the dye in the tumor with no accumulation in the normal tissues, the present results imply that the toxicity to normal tissues could be reduced significantly with selective destruction of the tumor following photodynamic treatment with the use of i.t. administration of specific antibodies conjugated to photosensitizers.

Animals↗
PubMed2003

Cellular uptake, localization and photodynamic effects of haematoporphyrin derivative in human glioma and squamous carcinoma cell lines.

Uptake, intracellular concentration, localization and photodynamic effects of a haematoporphyrin derivative (HpD, Photosan-3) were compared in human glioma (BMG-1, wild-type p53) and squamous carcinoma (4451, mutated p53) cell lines. Concentration and time dependence of cellular uptake of HpD was assayed from methanol extracts and whole cell suspension spectroscopy, while localization was studied by fluorescence microscopy-based image analysis. Colony-forming ability, apoptosis, cell-cycle progression and cytogenetic damage (micronuclei formation) were investigated as parameters of photodynamic response following irradiation with red light. BMG-1 cells were more sensitive to the photodynamic treatment than 4451 cells, although the 4451 cells accumulated a higher amount of HpD and did not differ significantly from BMG-1 cells with respect to intracellular localization. Photodynamically-induced cytogenetic damage and apoptosis were considerably higher in BMG-1 cells as compared to 4451 cells. The present results strongly suggest that manifestation of the photodynamically-induced lesions in the form of cytogenetic damage and apoptosis are among the important determinants of cellular sensitivity to HpD-PDT besides the photodynamic dose (intracellular concentration of the photosensitizer and the light dose).

Apoptosis↗
PubMed2003

cDNA cloning, expression and characterization of an allergenic L3 ribosomal protein of Aspergillus fumigatus.

Aspergillus fumigatus (Afu) is an important fungal pathogen causing allergic and invasive respiratory disorders. A plethora of multi-functional allergens/antigens secreted by Afu have been implicated in pathogenesis. The present study was undertaken to identify and characterize novel Afu allergen/antigen by cDNA library approach. cDNA library of Afu was immunoscreened with pooled sera of allergic bronchopulmonary aspergillosis (ABPA) patients. The cDNA clone, TS1, reacting significantly with specific IgG antibodies, was selected. cDNA was subcloned and expressed in Escherichia coli. Sequencing of the cDNA revealed an open reading frame (ORF) of 1179 bases coding for a protein with an approximate molecular weight of 44 kDa. Immunoreactivity of the recombinant TS1 protein (rTS1) was evaluated by ELISA and Western blot analysis using pooled sera of ABPA patients. The rTS1 exhibited binding to specific IgG and IgE antibodies present in sera of ABPA patients. The deduced amino acid sequence showed homology to 60S ribosomal protein L3 (RpL3) of Aspergillus nidulans, Saccharomyces cerevisiae and Homo sapiens. The RpL3 of S. cerevesiae, tcm1, to which TS1 sequence shows significant homology (72% identity), is known to be responsible for conferring resistance against trichodermin (antibiotic, inhibiting protein synthesis). The present study has led to identification, cloning and expression of a 44-kDa novel allergen/antigen of Afu with sequence homology to L3 ribosomal protein with a probable role in resistance of Afu to antifungal drugs. Sixty-four per cent sequence identity of Afu RpL3 with human RpL3 and common regions in their predicted epitopes suggest a possibility of involvement of Afu RpL3 in autoimmune reactions due to molecular mimicry.

Algorithms↗
PubMed2003

Role of apoptosis in photodynamic sensitivity of human tumour cell lines.

Photodynamic therapy (PDT) using a photosensitizer, such as haematoporphyrin derivative (HpD), in conjunction with visible light is a promising new modality to treat localized cancer. Cell death caused by PDT (through the generation of reactive oxygen species) can occur either by apoptosis (interphase death or as a secondary event following mitosis) and/or necrosis depending on the cell type, concentration and intracellular localization of the sensitizer, and the light dose. Since, apoptosis induced by PDT treatment plays an important role in determining the photodynamic efficacy, in the present work we have investigated the role of apoptotic cell death in relation to the observed differences in sensitivity to HpD-PDT between a human glioma cell line (BMG-1) carrying wild-type tumour suppressor gene p53 and a human squamous carcinoma cell line (4451) with mutated p53. HpD (photosan-3; PS-3) -PDT induced apoptosis was studied by: [A] flow-cytometric analysis of DNA content (sub G0/G1 population); [B] phosphatidylserine externalization (Annexin-V +ve cells); [C] cell size and cytoskeleton reorganization (light-scatter analysis); and [D] fluorescence microscopy (morphological features). PS-3-PDT induced a significantly higher level of apoptosis in BMG-1 cells as compared to 4451 cells. This was dependent on the concentration of PS-3 as well as post-irradiation time in both the cell lines. At 2.5 microg/ml of PS-3 the fraction of BMG-1 cells undergoing apoptosis (60%) was nearly 6 folds higher than 4451 cells (10%). In BMG-1 cells the induction of apoptosis increased with PS-3 concentration up to 5 microg/ml (>80%). However, a decrease was observed at a concentration of 10 microg/ml, possibly due to a shift in the mode of cell death from apoptosis to necrosis. In 4451 cells, on the other hand, the increase in apoptosis could be observed even up to 10 microg/ml of PS-3 (60%). Present results show that the higher sensitivity to PS-3-PDT in glioma cells arise on account of a higher level of apoptosis and suggest that induction of apoptosis is an important determinant of photodynamic sensitivity in certain cell types.

Apoptosis↗
PubMed2002

Thermostable alpha-amylase conjugated antibodies as probes for immunodetection in ELISA.

Thermostable alpha-amylase from B. licheniformis has been conjugated with high efficiency to goat antibodies against human, mouse, and rabbit immunoglobulins to prepare second-step reagents which can be used in Enzyme Linked Immunosorbent Assays (ELISA). Various conjugation methods, such as one- and two-step glutaraldehyde coupling and cross-linking, using heterobifunctional reagents such as sulfosuccinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carbonate (sulfo-SMCC) and N-succinimidyl-S-acetylthioacetate (SATA), yielded active alpha-amylase labeled second antibodies. Such conjugates had molecular sizes ranging between 200-300 kDa. Filter sterilized solutions of conjugates, when stored at 37 degrees C for two weeks, retained 32% of their biological activity and were thermostable even after keeping for 1 h at 90 degrees C.

Animals↗
PubMed2001

Ventilation strategy for video-assisted thoracoscopic clipping of patent ductus arteriosus in children.

Video-assisted endoscopic techniques have reduced operative trauma in adult thoracic and general surgery but its application in children with congenital heart disease has been limited. We report the use of video-assisted thoracoscopic (VAT) technique of clipping patent ductus arteriosus (PDA) in children. Forty patients with PDA were divided into two groups: during VAT surgery patients in group A [mean age=3.6 +/- 2.4 (SD) years] were managed with right main stem bronchial intubation and those in the group B [mean age=3.7 +/- 2.7 (SD) years] received low tidal volume-high frequency ventilation using a Siemens 900C ventilator. The mean oxygen saturation (SpO2) observed during the surgical intervention was significantly lower in group A (90%) compared to group B (96.8%) while the surgical convenience was not different. We conclude that a low tidal volume-high frequency ventilation is acceptable and safe in patients with PDA undergoing VATS.

Bronchi↗
PubMed2001

Equilibrium denaturation of buffalo pituitary growth hormone.

To understand the structural properties of buffalo growth hormone (buGH), the equilibrium denaturation using guanidinium chloride (GdmCl) was carried out and was monitored by ultraviolet absorption spectroscopy, intrinsic fluorescence spectroscopy, far UV-circular dichroism and size-exclusion chromatography. The normalized denaturation transition curves for each of the above methods were not coincident, showing that buGH does not follow a simple two state folding mechanism. Further, size-exclusion chromatography also showed the presence of an associated intermediate during the unfolding of buGH. It was observed that in buGH, denaturation resulted in an initial disruption of the tertiary structure, whereas the secondary structure and the degree of compactness were disrupted at a higher concentration of the denaturant. This suggests that buGH follows the hierarchical model of protein folding.

Animals↗
PubMed2001

Physico-chemical characterization of growth hormone from water buffaloes (Bubalus bubalis).

A purified preparation of growth hormone from pituitaries of water buffaloes (Bubalus bubalis) has been extensively characterized with regard to physico-chemical properties. The molecular size of buffalo GH (buGH) by electrospray ionization mass spectroscopy (ES-MS) was found to be 21394.00+/-8.44Da and its stokes radius was determined as 2.3 nm. Size heterogeneity in buffalo GH was checked both by electrophoresis and molecular sieve chromatography using 125I-labelled buffalo GH. Similar size heterogeneity was found in standard preparations of ovine and bovine growth hormones. Isoelectric focussing and chromatofocussing indicated charge heterogeneity in buffalo GH preparation. Major charge isoforms having pI of 7.2, 7.7 and minor forms in the pI range of 5.7 to 7.0 were found. Lectin chromatography on Concanavalin A matrix showed that less than 1% of buffalo GH was glycosylated. Heterogeneity in NH2-terminal sequence was also observed, with alanine, phenylalanine and methionine as the NH2-terminal residues as checked by dansyl and DABITC methods. Estimation of tryptophan residue indicated that a single tryptophan residue was present. Ellman's method showed presence of two disulfide bridges per mole of buffalo GH. Intrinsic fluorescence spectrum of buffalo GH exhibited lambda emission maximum at 337 nm. UV-CD spectrum showed that almost 48% of the secondary structure of buGH was constituted by alpha-helicity. The T(M) of buGH as determined by differential scanning calorimetric (DSC) studies was found to be 63 degrees C.

Animals↗